The translational control of protein synthesis by hemin in rabbit reticulocytes is mediated by the formation of a high molecular weight protein inhibitor of polypeptide chain initiation termed the hemin-controlled translational repressor (HCR). HCR acts as a specific protein kinase by phosphorylating the initiation factor (eIF-2) that mediates binding of the initiator tRNA (met-tRNAf) to 40 s ribosomal subunits. We plan to determine the precise molecular effect(s) the phosphorylation of eIF-2 has on polypeptide chain initiation. The effect of HCR on polypeptide chain initiation will be studied in the cryde lysate using (35S)Met-tRNAf and (35S)fMet-tRNAf as well as radiolabeled, purified, globin mRNA and eIF-2 as probes. The distribution of endogenous eIF-2 under various experimental conditions will be measured by using a radioimmunoassay for this factor, that is to be developed. The role that deacylation of ribosome-bound Met-tRNAf may play in the regulation by HCR will be explored by comparing the effects of HCR on the subunit labeling by formylated and non-formylated Met-tRNAf. The ribosomal Met-tRNAf hydrolase that may mediate this effect will be isolated and characterized. We also intend to isolate and characterize the phosphatase in reticulocyte lysate that dephosphorylates eIF-2.